Insulin Receptor Signaling in the GnRH Neuron Plays a Role in the Abnormal GnRH Pulsatility of Obese Female Mice

Infertility associated with obesity is characterized by abnormal hormone release from reproductive tissues in the hypothalamus, pituitary, and ovary. These tissues maintain insulin sensitivity upon peripheral insulin resistance. Insulin receptor signaling may play a role in the dysregulation of gonadotropin-releasing hormone (GnRH) secretion in obesity, but the interdependence of hormone secretion in the reproductive axis and the multi-hormone and tissue dysfunction in obesity hinders investigations of putative contributing factors to the disrupted GnRH secretion. To determine the role of GnRH insulin receptor signaling in the dysregulation of GnRH secretion in obesity, we created murine models of diet-induced obesity (DIO) with and without intact insulin signaling in the GnRH neuron. Obese control female mice were infertile with higher luteinizing hormone levels and higher GnRH pulse amplitude and total pulsatile secretion compared to lean control mice. In contrast, DIO mice with a GnRH specific knockout of insulin receptor had improved fertility, luteinizing hormone levels approaching lean mice, and GnRH pulse amplitude and total secretion similar to lean mice. Pituitary responsiveness was similar between genotypes. These results suggest that in the obese state, insulin receptor signaling in GnRH neurons increases GnRH pulsatile secretion and consequent LH secretion, contributing to reproductive dysfunction.     

Mammalian gonadotropin-releasing hormone (GnRH) receptor subtypes

Mammalian gonadotropin-releasing hormone (GnRH I) is a hypothalamic decapeptide that governs gonadotropin secretion through interaction with its seven transmembrane (7TM), G protein-coupled receptor (GPCR) expressed by anterior pituitary cells. A second decapeptide, GnRH II, originally discovered in the chicken hypothalamus was recently reported to be expressed in the mammalian hypothalamus as well. A search of the recently-sequenced human genome identified a 7TM/GPCR on chromosome 1 that exhibited a higher identity with non-mammalian vertebrate GnRH II receptors (55%) than with the human GnRH I receptor (39%). Molecular cloning and nucleotide sequencing of this putative GnRH II receptor cDNA from monkey pituitary gland revealed a 379 amino acid receptor that, unlike the GnRH I receptor, possessed a C-terminal tail. Heterologous expression and functional testing of the receptor in COS-1 cells confirmed its identity as a GnRH II receptor: measurement of 3H-inositol phosphate accumulation revealed EC(50)s for GnRH II of 0.86 nM and for GnRH I of 337 nM. Ubiquitous tissue expression of GnRH II receptor mRNA was observed using a human tissue RNA expression array and a 32P-labeled antisense riboprobe representing the 7TM region of human GnRH II receptor cDNA. As predicted by the presence of its C-terminal tail, the GnRH II receptor was desensitized by GnRH II treatment whereas the naturally tail-less GnRH I receptor was not desensitized by GnRH I. Pharmacological analysis of the GnRH II receptor revealed that GnRH I 'superagonists' were more potent than GnRH I but less potent than GnRH II. Numerous GnRH I antagonists showed neither antagonistic nor agonistic activity with the GnRH II receptor. The functions of the GnRH II receptor are unknown but may include regulation of gonadotropin secretion, female sexual behavior, or tumor cell growth.     

Interplay of KNDy and nNOS neurons: A new possible mechanism of GnRH secretion in the adult brain

Reproduction in mammals is favoured when there is sufficient energy available to permit the survival of offspring. Neuronal nitric oxide synthase expressing neurons produce nitric oxide in the proximity of the gonadotropin-releasing hormone neurons in the preoptic region. nNOS neurons are an integral part of the neuronal network controlling ovarian cyclicity and ovulation. Nitric oxide can directly regulate the activity of the GnRH neurons and play a vital role neuroendocrine axis. Kisspeptin neurons are essential for the GnRH pulse and surge generation. The anteroventral periventricular nucleus (AVPV), kisspeptin neurons are essential for GnRH surge generation. KNDy neurons are present in the hypothalamus's arcuate nucleus (ARC), co-express NKB and dynorphin, essential for GnRH pulse generation. Kisspeptin-neurokinin B-dynorphin (KNDy) neuroendocrine molecules of the hypothalamus are key components in the central control of GnRH secretion. The hypothalamic neurons kisspeptin, KNDy, nitric oxide synthase (NOS), and other mediators such as leptin, adiponectin, and ghrelin, play an active role in attaining puberty. Kisspeptin signalling is mediated by NOS, which further results in the secretion of GnRH. Neuronal nitric oxide is critical for attaining puberty, but its direct role in adult GnRH secretion is poorly understood. This review mainly focuses on the role of nNOS and its interplay with KNDy neurons in the hormonal regulation of reproduction.     

Progesterone stimulation of in vitro GnRH release from the hypothalamus of the estrogen-primed, ovariectomized rat: serotoninergic role

The ability of ovarian steroids to affect luteinizing hormone secretion is closely related to the influence of these steroids on the activities of several neurotransmitter systems within specific areas of the hypothalamus and associated brain areas. The purpose of this study was to characterize in vitro progestagenic effects on serotonin (5-hydroxytryptamine, 5-HT) and gonadotropin-releasing hormone (GnRH) release from hypothalamic slices from estrogen-primed, ovariectomized rats. Results of this study show that (1) progesterone can stimulate in vitro GnRH and 5-HT release from hypothalamic tissue slices of ovariectomized rats primed with estrogen and (2) the 5-HT receptor antagonist mianserin blocks the ability of progesterone to augment in vitro GnRH release from these tissue slices. This suggests that the influence of progesterone on the estrogen-induced LH surge is, at least in part, via progestagenic release of 5-HT and the subsequent effect of this neurotransmitter on the release of GnRH within the hypothalamus.     

Calcium receptor stimulates chemotaxis and secretion of MCP-1 in GnRH neurons in vitro: potential impact on reduced GnRH neuron population in CaR-null mice

The factors controlling the migration of mammalian gonadotropin-releasing hormone (GnRH) neurons from the nasal placode to the hypothalamus are not well understood. We studied whether the extracellular calcium-sensing receptor (CaR) promotes migration/chemotaxis of GnRH neurons. We demonstrated expression of CaR in GnRH neurons in the murine basal forebrain and in two GnRH neuronal cell lines: GT1-7 (hypothalamus derived) and GN11 (olfactory bulb derived). Elevated extracellular Ca(2+) concentrations promoted chemotaxis of both cell types, with a greater effect in GN11 cells. This effect was CaR mediated, as, in both cell types, overexpression of a dominant-negative CaR attenuated high Ca(2+)-stimulated chemotaxis. We also demonstrated expression of a beta-chemokine, monocyte chemoattractant protein-1 (MCP-1), and its receptor, CC motif receptor-2 (CCR2), in the hypothalamic GnRH neurons as well as in GT1-7 and GN11 cells. Exogenous MCP-1 stimulated chemotaxis of both cell lines in a dose-dependent fashion; the effect was greater in GN11 than in GT1-7 cells, consistent with the higher CCR2 mRNA levels in GN11 cells. Activating the CaR stimulated MCP-1 secretion in GT1-7 but not in GN11 cells. MCP-1 secreted in response to CaR stimulation is biologically active, as conditioned medium from GT1-7 cells treated with high Ca(2+) promoted chemotaxis of GN11 cells, and this effect was partially attenuated by a neutralizing antibody to MCP-1. Finally, in the preoptic area of anterior hypothalamus, the number of GnRH neurons was approximately 27% lower in CaR-null mice than in mice expressing the CaR gene. We conclude that the CaR may be a novel regulator of GnRH neuronal migration likely involving, in part, MCP-1.     

Molecular integration of hypothalamo-pituitary-adrenal axis-related neurohormones on the GnRH neuron.

Gonadotropin-releasing hormone (GnRH) secretion from the hypothalamus is pivotal to the regulation of reproductive physiology in vertebrates. GnRH and the reproductive axis, in general, can be inhibited during periods of stress or injury. Stress, in the form of mechanical, psychological or immunological insult to an organism results in the activation of the hypothalamo-pituitary-adrenal (HPA) axis initiated by the hypothalamic release of corticotropin-releasing factor (CRF). Recent studies indicate that CRF may act either directly on the GnRH neuron to down-regulate GnRH synthesis, or indirectly via a beta-endorphin-mediated pathway. Moreover, in vitro studies suggest that CRF-related peptides can increase the sensitivity of the GnRH neuron to prolactin by increasing the synthesis of the prolactin receptor.     

促性腺激素释放激素的结构及其生物学功能

促性腺激素释放激素(GnRH)是下丘脑分泌的十肽激素,是神经、免疫、内分泌三大调节系统互相联系的重要信号分子,对生殖调控具有重要意义.GnRH类似物是近年来应用最广的多肽类激素新药之一.就GnRH及其受体的结构及分布、GnRH在垂体和性腺水平调控生殖的一系列证据、影响GnRH释放的因素等进行了综述,并展望了GnRH研究的发展趋势及应用前景.     

Effects of GABA(A) receptor modulation on the expression of GnRH gene and GnRH receptor (GnRH-R) gene in the hypothalamus and GnRH-R gene in the anterior pituitary gland of follicular-phase ewes

The effect of prolonged, intermittent infusion of GABA(A) receptor agonist (muscimol) or GABA(A) receptor antagonist (bicuculline) into the third cerebral ventricle on the expression of GnRH gene and GnRH-R gene in the hypothalamus and GnRH-R gene in the anterior pituitary gland was examined in follicular-phase ewes by real-time PCR. The activation or inhibition of GABA(A) receptors in the hypothalamus decreased or increased the expression of GnRH and GnRH-R genes and LH secretion, respectively. The present results indicate that the GABAergic system in the hypothalamus of follicular-phase ewes may suppress, via hypothalamic GABA(A) receptors, the expression of GnRH and GnRH-R genes in this structure. The decrease or increase of GnRH-R mRNA in the anterior pituitary gland and LH secretion in the muscimol- or bicuculline-treated ewes, respectively, is probably a consequence of parallel changes in the release of GnRH from the hypothalamus activating GnRH-R gene expression. It is suggested that GABA acting through the GABA(A) receptor mechanism on the expression of GnRH gene and GnRH-R gene in the hypothalamus may be involved in two processes: the biosynthesis of GnRH and the release of this neurohormone in the hypothalamus.     

Gonadotropin secretion in ovariectomized ewes: effect of passive immunization against gonadotropin-releasing hormone (GnRH) and infusion of a GnRH agonist and estradiol

Gonadotropin secretion was examined in ovariectomized sheep after passive immunization against gonadotropin-releasing hormone (GnRH). Infusion of ovine anti-GnRH serum, but not control antiserum, rapidly depressed serum concentrations of luteinizing hormone (LH). The anti-GnRH-induced reduction in serum LH was reversed by circhoral (hourly) administration of a GnRH agonist that did not cross-react with the anti-GnRH serum. In contrast, passive immunization against GnRH led to only a modest reduction in serum concentrations of follicle-stimulating hormone (FSH). Pulsatile delivery of the GnRH agonist did not influence serum concentrations of FSH. Continuous infusion of estradiol inhibited and then stimulated gonadotropin secretion in animals passively immunized against GnRH, with gonadotrope function driven by GnRH agonist. However, the magnitude of the positive feedback response was only 10% of the response noted in controls. These data indicate that the estradiol-induced surge of LH secretion in ovariectomized sheep is the product of estrogenic action at both hypothalamic and pituitary loci. Replacement of the endogenous GnRH pulse generator with an exogenous generator of GnRH-like pulses that were invariant in frequency and amplitude could not fully reestablish the preovulatory-like surge of LH induced by estradiol.     

Expression of the GnRH and GnRH receptor (GnRH-R) genes in the hypothalamus and of the GnRH-R gene in the anterior pituitary gland of anestrous and luteal phase ewes

Data exists showing that seasonal changes in the innervations of GnRH cells in the hypothalamus and functions of some neural systems affecting GnRH neurons are associated with GnRH release in ewes. Consequently, we put the question as to how the expression of GnRH gene and GnRH-R gene in the hypothalamus and GnRH-R gene in the anterior pituitary gland is reflected with LH secretion in anestrous and luteal phase ewes. Analysis of GnRH gene expression by RT-PCR in anestrous ewes indicated comparable levels of GnRH mRNA in the preoptic area, anterior and ventromedial hypothalamus. GnRH-R mRNA at different concentrations was found throughout the preoptic area, anterior and ventromedial hypothalamus, stalk/median eminence and in the anterior pituitary gland. The highest GnRH-R mRNA levels were detected in the stalk/median eminence and in the anterior pituitary gland.During the luteal phase of the estrous cycle in ewes, the levels of GnRH mRNA and GnRH-R mRNA in all structures were significantly higher than in anestrous ewes. Also LH concentrations in blood plasma of luteal phase ewes were significantly higher than those of anestrous ewes.In conclusion, results from this study suggest that low expression of the GnRH and GnRH-R genes in the hypothalamus and of the GnRH-R gene in the anterior pituitary gland, amongst others, may be responsible for a decrease in LH secretion and the anovulatory state in ewes during the long photoperiod.     

Synthesis and secretion of GnRH

Comprehensive studies have provided a clear understanding of the effects of gonadal steroids on the secretion of gonadotropin releasing hormone (GnRH), but some inconsistent results exist with regard to effects on synthesis. It is clear that regulation of both synthesis and the secretion of GnRH are effected by neurotransmitter systems in the brain. Thus, steroid regulation of GnRH synthesis and secretion can be direct, but the predominant effects are transmitted through steroid-responsive neuronal systems in various parts of the brain. There is also emerging evidence of direct effects on GnRH cells. Overriding effects on synthesis and secretion of GnRH can be observed during aging, in undernutrition and under stressful situations; these involve various neuronal systems, which may have serial or parallel effects on GnRH cells. The effect of aging is accompanied by changes in GnRH synthesis, but comprehensive studies of synthesis during undernutrition and stress are less well documented. Altered GnRH and gonadotropin secretion that occurs in seasonal breeding animals and during the pubertal transition is not generally accompanied by changes in GnRH synthesis. Secretion of GnRH from the brain is a reflection of the inherent function of GnRH cells and the inputs that integrate all of the central regulatory elements. Ultimately, the pattern of secretion dictates the reproductive status of the organism. In order to fully understand the central mechanisms that control reproduction, more extensive studies are required on the neuronal circuitry that provides input to GnRH cells.     

Regulation of gonadotropin-releasing hormone (GnRH) gene expression in hypothalamic neuronal cells

Summary 1. Gonadotropin-releasing hormone (GnRH) is the hypothalamic releasing factor that controls pituitary gonadotropin subunit gene expression and indirectly gametogenesis and steroidogenesis from the gonad, which results in reproductive competence.2. GnRH is synthesized in only about 1000 neurons in the hypothalamus and released in an episodic fashion down the median eminence to regulate gonadotropin biosynthesis.3. Although much is known about the secretory dynamics of GnRH release, little is known about the pretranslational control of GnRH biosynthesis due to lack of appropriate model systems. The recent availability of immortalized neuronal cell lines that produce GnRH allows investigators for the first time to begin to dissect the factors that directly regulate GnRH gene expression.4. This article reviews the current state of knowledge concerning the mechanisms that direct tissue-specific and peptide hormone control of GnRH biosynthesis.     

GnRH and GnRH receptors: distribution,function and evolution

Gonadotropin‐releasing hormone (GnRH) was originally identified because of its essential role in regulating reproduction in all vertebrates. Since then, three phylogenetically related GnRH decapeptides have been characterized in vertebrates and invertebrates. Almost all tetrapods investigated have at least two GnRH forms (GnRH1 and GnRH2) in the central nervous system. From distributional and functional studies in vertebrates, GnRH1 in the hypothalamus projects predominantly to the pituitary and regulates reproduction via gonadotropin release. GnRH2, which is located in the midbrain, projects to the whole brain and is thought to be involved in sexual behaviour and food intake. GnRH3, located in the forebrain, has only been found in teleost fish and appears to be involved in sexual behaviour, as well as, in some fish species, gonadotropin release. Multiple GnRH receptors (GnRH‐Rs), G‐protein‐coupled receptors regulate endocrine functions and neural transmissions in vertebrates. Phylogenetic and structural analyses of coding sequences show that all vertebrate GnRH‐Rs cluster into two main receptor types comprised of four subfamilies. This suggests that at least two rounds of GnRH receptor gene duplications may have occurred in different groups within each lineage. Functional studies suggest that two particular subfamilies of GnRH receptors have independently evolved to act as species‐specific endocrine modulators in the pituitary, and these show the greatest variety in regulating neuron networks in the brain. Given the long evolutionary history of the GnRH system, it seems likely that much more remains to be understood about its roles in behaviour and function of vertebrates.     

The hypogonadotropic state of the prepubertal male rhesus monkey (Macaca mulatta) is not associated with a decrease in hypothalamic gonadotropin-releasing hormone content

Hypothalamic contents of gonadotropin-releasing hormone (GnRH) in neonatally orchidectomized infant, juvenile, and adult monkeys were measured by a radioimmunoassay (RIA) and by an in vivo bioassay that utilized luteinizing hormone (LH) secretion in estrogen- and progesterone-treated ovariectomized rats. The results of the bioassay provided no evidence to suggest that hypothalamic GnRH content in juvenile monkeys (mean = 83 ng/hypothalamus; n = 3) was less than that in infants (mean = 54 ng/hypothalamus; n = 4) and adults (mean = 36 ng/hypothalamus; n = 3). A similar developmental pattern in hypothalamic GnRH content was also observed when the decapeptide was measured by RIA. In striking contrast to the maintenance of hypothalamic GnRH content throughout postnatal development, pituitary gonadotropin contents and serum gonadotropin concentrations were markedly reduced in juvenile monkeys.     

Differential actions of GnRH and rat hypothalamic extracts in release of hypophysial FSH and LH in vitro.

A study was made of the separate patterns of luteinizing hormone (LH) and follicle stimulating hormone (FSH) release from isolated rat pituitary tissue evoked by synthetic gonadotropin releasing hormone (GnRH) or female hypothalamic extracts (HE), respectively, in a continuous perifusion system. Under defined conditions, gonadotropin release from hemipituitaries was relatively stable and reproducible. Absolute levels of LH and FSH release evoked by HE in terms of their GnRH content were always greater than those following exposure to synthetic GnRH at varying doses. Synthetic GnRH released more FSH than LH. In contrast, the HE released slightly higher levels of LH than FSH. The data suggest that the female rat hypothalamus contains substances other than GnRH, capable of releasing both LH and FSH. It is possible that such unidentified components can modify the hypophysial action of GnRH, resulting in particular circumstances in a differential release of LH and FSH.     

Gonadotropin-releasing hormone (GnRH) receptor dynamics and gonadotrope responsiveness during and after continuous GnRH stimulation

Gonadotrope responsiveness, serum and tissue levels of luteinizing hormone (LH), and tissue concentration of gonadotropin-releasing hormone (GnRH) receptors in ovariectomized rats were determined during and after continuous GnRH stimulation. Intraperitoneal placement of GnRH-containing osmotic minipumps for 96 h established a rate of GnRH delivery (1 microgram/h) that resulted in stable serum levels of GnRH (500-700 pg/ml). Secretion of LH increased 8-fold within 6 h; however, serum LH returned to pretreatment levels by 24 h, even with continued GnRH stimulation. Tissue concentration of LH was depressed within 48 h of initiation of treatment but levels were restored by 96 h. Tissue levels of GnRH receptor remained elevated during the first 6 h of treatment but were reduced by 60% within 24 h and remained depressed for the duration of treatment. Gonadotrope responsiveness 48 h and 96 h after initiation of treatment was reduced by 50% and 90%, respectively. Removal of the GnRH delivery vehicle resulted in rapid disappearance of GnRH from serum. Dramatic reduction (75%) in circulating levels of LH, and a 2-fold increase in tissue levels of LH and in GnRH receptor concentration were noted within 6 h of minipump removal. Although tissue concentration of GnRH receptor returned to pretreatment levels within 48 h of minipump removal, both basal LH secretion and gonadotrope responsiveness remained depressed even 96 h after cessation of continuous GnRH stimulation. These data indicate that GnRH can "down regulate" its receptor, gonadotrope responsiveness is not obligatorily linked to receptor concentration, and desensitization that follows hyperstimulation represents effects directed at post-receptor loci.